PROJECT SUMMARY Aging and cancer are inextricably linked. Genome instability is common to both aging and cancer, whereby genetic instability and cancer risk increase with age. However, there is a fundamental gap in understanding the mechanisms that predispose us to increased genetic instability as we age. Thus, the overall objective of this application is to fill this gap in knowledge. Mutations are not random, and there are endogenous mutation ?hotspot? regions of the genome. Importantly, many of these cancer-assoicated mutation hotspots co-localize with, and are enriched at sequences capable of adopting alternatively structured DNA (i.e. non-B DNA), implicating these sequences in cancer etiology. We have discovered that naturally occurring non-B DNA; e.g., Z-DNA and H-DNA, is mutagenic in mammals, and this DNA structure-induced mutation increases with age. However, the mechanisms involved in the age-related propagation of genetic instability at these hotspots are unclear. Thus, a goal is to determine the mechanisms involved in DNA structure-induced genetic instability, and the impact of increased age on the generation of these endogenous mutation hotspots. We have also found that non-B DNA is processed by DNA repair and replication proteins. These findings provide the basis for the scientific premise going forward to test the novel hypothesis that cancer relevant DNA structure-induced genetic instability increases with age, because with age, non-B DNA (i.e. an endogenous mutation hotspot) becomes increasingly refractory to repair; moreover, non-B DNA is subject to error-prone processing that increases with age. Both mechanisms lead to the generation of age-dependent mutations. The long-term goals are to elucidate the impact of aging on genetic instability at mutation hotspots, to determine the mechanisms of age-related, and tissue-specific DNA structure-induced genetic instability in cancer etiology, and to develop novel approaches to reduce genetic instability to prevent and/or treat age-related disease. The objectives are to determine the role(s) of age in DNA structure-induced genomic instability in specific tissues of mice, and to identify the mechanisms of replication-dependent and -independent genetic instability. We will use novel mutation-reporter mice that we have developed to determine the effects of age on the mutagenic potential of non-B DNA sequences, and determine the mechanisms of the processing of these mutagenic sequences. This is innovative because it will test the novel hypothesis that DNA structure-induced genetic instability increases with age in mammals, and that these structure-forming sequences are processed by DNA repair mechanisms whose efficiency decline with age. The expected contribution is the elucidation of the impact of age on genetic instability and of the mechanisms involved in the generation of endogeous mutation hotspots in cancer, to provide a better understanding of age-related disease etiology. This is significant because the results will aid in the development of new strategies to mitigate age-related diseases.